1. Field of the Invention
This invention relates to a method for the kinetic determination of cholesterol using a cholesterol dehydrogenase as well as to a reagent composition therefor. More particularly, the present invention relates to a method for the determination of cholesterol which comprises incubating a sample, a cholesterol dehydrogenase, and nicotinamide-adenine dinucleotide (after referred to as NAD) or nicotinamide-adenine dinucleotide phosphate (after referred to as NADP) in the presence of from about 10 to about 100 mg/ml of a surfactant and then measuring the resulting detectable change kinetically, as well as to a reagent composition therefor.
2. Description of the Prior Art
As enzymatic methods for the determination of cholesterol, there are known methods wherein free cholesterol and esterified cholesterol are subjected to chemical or enzymatic saponification to convert the latter cholesterol to free cholesterol. All the free cholesterols are allowed to react with a cholesterol oxidase, and the formed hydrogen peroxide or cholestenone or the consumed oxygen is measured (Clin. Chem., 20, 470, 1974; U.S. Pat. Nos. 3,925,164 and 4,212,938 and GB Pat. No. 1,412,244). The most widely used of these methods using a cholesterol oxidase is a method wherein the formed hydrogen peroxide is allowed to react with a peroxidase and a color-producing reagent and the resulting colored substance is measured. However, this method has drawbacks in that a reagent of intricate composition needs to be used and the measurement is affected by bilirubin and ascorbic acid both present in blood together with cholesterol causing a measurement error.
There are also known methods wherein, in place of the cholesterol oxidase used above, a cholesterol dehydrogenase and NAD or NADP as a connzyme are used and the formed cholestenone or the reduced type NAD (after referred to as NADH) or reduced type NADP (after referred to as NADPH) formed is measured (U.S. Pat. No. 4,181,575; FRG Patent Laid-open No. 3,032,377 and Japanese Patent Laid-open No. 89,200/1983). Of these methods using a cholesterol dehydrogenase, the method of measuring the formed NADH or NADPH is advantageous in that the measurement is not affected by the above mentioned hindering substances present in blood together with cholesterol.
These conventional methods for the determination of cholesterol using a cholesterol oxidase or a cholesterol dehydrogenase are so-called end point assay methods and, in these methods, cholesterol as a substrate must be allowed to react until it is completely converted to a reaction product. Therefore, there has generally been employed a measurement time of 5 to 10 min, a blank test for each sample and a relatively large amount of an enzyme. In recent years, in the field of clinical chemical inspection, measuring a large number of samples in a short time and with accuracy has been required which has led to the development of automatic analytical equipment and apparatuses. In measurements by automatic analytical equipment and apparatuses, the measurement time is required to be as short as possible. Hence, in place of the end point assay methods, there were proposed methods wherein the initial rate of reaction is measured, namely, kinetic measurement methods called "rate assay". In the study on method for the determination of the cholesterol, too, there was tried a kinetic measurement method using a cholesterol oxidase. However, the reaction did not proceed according to the first order or pseudo-first order because the Km (Michaelis's constant) value of the enzyme was too low compared with 500.0 mg/dl or more of cholesterol (this is a level necessary for determination of cholesterol). It is reported that, in order to artificially increase this unacceptably low Km value, a method of adding 3,4-dichlorophenol was tried and, as a result, the Km value of cholesterol oxidase was increased and kinetic measurement of cholesterol has been made possible (European Pat. No. 53,692). However, this method of using a cholesterol oxidase is not free from the above mentioned interference by hindering substances present in blood.